A Nutritionally Meaningful Increase in Vitamin D in Retail Mushrooms Is

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J Journal of Nutrition & Food Sciences DOI: 10.4172/2155-9600.1000236 ISSN: 2155-9600

Research Article Open Access A Nutritionally Meaningful Increase in Vitamin D in Retail Mushrooms is Attainable by Exposure to Sunlight Prior to Consumption Katherine M Phillips* and Amy S Rasor Research Scientist, Biochemistry Department, Virginia Tech, USA

Abstract

The vitamin D2 content of white button mushrooms is relatively low. UV exposure produces vitamin D2 by rapid conversion of ergosterol to ergocalciferol. Commercial-scale UV treatment has been used to produce vitamin

D-enhanced mushrooms. The reliability of a consumer-friendly protocol to increase vitamin D2 in mushrooms by a nutritionally meaningful amount using exposure to sunlight was evaluated. Sliced white button mushrooms were exposed to sunlight for 15, 30, or 60 minutes in 16 experiments at different times of day, seasons, and cloud cover.

Vitamin D2 was measured by HPLC with 3H-vitamin D3 internal standard. Change in vitamin D2 per 70 g serving

relative to untreated mushrooms was evaluated. Vitamin D2 in all unexposed mushrooms was <30 IU/70 g (<5% DRI) (median, <7 IU/70 g). Regardless of season, treatment for 15 minutes between 9:30 a.m. and 3:30 p.m. under

partly cloudy to clear conditions increased vitamin D2 by157-754 IU/70 g (26-126%DRI), and up to 1142 IU/70 g total increase was observed after 30 min. On overcast and mostly cloudy days the gain was 76-178 IU/70 g (13-30%DRI) after 15 minutes, but after one hour the level was comparable to 15 minutes of treatment in clear conditions. Trials by consumers at four different geographic locations resulted in increases of 367-905 IU/70 g. A preliminary trial showed

dramatically elevated vitamin D2 contents in other mushroom types exposed 15 minutes under clear conditions.

These results demonstrate that vitamin D2 in mushrooms can be reliably enhanced by at least 25% of the DRI (150 IU; 3.75 µg)/70 g serving by exposure to sunlight for as little as 15 minutes on a clear or partly cloudy day between 9:30 a.m. and 3:30 p.m., and >100% (>600 IU) in many cases. Even under conditions of lower UV intensity similar increases can be achieved after 30-60 minutes.

Keywords: Vitamin D; Sun; Ultraviolet light; irradiation; Nutrient and fortified foods are one means of delivery. In the U.S., fluid milk has intake; Dietary supplements; Food composition; Agaricus bisporus; been fortified with vitamin D since the 1930s [9], but more recently Shiitake; Oyster; Enoki other foods have been approved for fortification, including breakfast cereal, orange juice, processed cheese, yogurt, margarine, and ice

Introduction cream [27,28]. Most foods are fortified with vitamin D3, but vitamin D (ergocalciferol) is frequently used in dietary supplements and While the role of vitamin D in bone health has long been 2 vegetarian products such as soy milk. recognized, recent attention has focused on its role in immune function and prevention of cancer and other diseases [1-13]. Vitamin D2 is produced commercially by irradiation of yeast with Vitamin D3 (cholecalciferol) is produced naturally in humans from ultraviolet light (UV), which converts the fungal sterol ergosterol to 7-dehydrocholesterol in skin exposed to ultraviolet light, but many ergocalciferol in a process chemically analogous to the production individuals receive insufficient sun exposure to maintain adequate of vitamin D3 from 7-dehydrocholesterol [5,29,30]. The metabolism vitamin D status [5,14,15]. Living at latitudes >37°, vigilant use of vitamin D2 to its biologically active form, 1,25-dihydroxyvitamin of sunscreen, dark skin color, minimal time spent outdoors, low D2, and the effectiveness of vitamin D2 from supplements and edible consumption of vitamin D-rich foods and supplements, and age- mushrooms in improving vitamin D status has been reported [31-36]. related decline in the ability to synthesize the active form contribute Ergosterol occurs in milligram amounts in mushrooms, but microgram to less than optimal vitamin D levels [5]. The serum concentration of amounts of vitamin D2 have biological activity (1 µg =40 IU), so

25-hydroxyvitamin D, a metabolite of vitamin D that increases with sun conversion of a relatively small amount of ergosterol to vitamin D2 exposure and vitamin D intake, is generally accepted as an indicator of results in a nutritionally significant increase in vitamin D. vitamin D status. It is estimated that 30-50% of individuals in Europe All mushrooms contain ergosterol but the vitamin D2 content varies and the U.S. have insufficient levels [16-18]. Comprehensive reviews on widely. A recent study [37] found that in edible varieties of mushrooms the synthesis and metabolism of vitamin D are available [e.g., [5,19]]. The Food and Nutrition Board of the Institute of Medicine has established the Dietary Reference Intake (DRI) of 600 IU/day (15 µg) *Corresponding author: Katherine M Phillips, Research Scientist, 304 Engel Hall, for children and adults up to age 70, and 800 IU (20 µg) for individuals Biochemistry Department (0308), Virginia Tech, Blacksburg, VA 24061, USA, Tel: >70 years of age [20,21]. The revised DRIs are a substantial increase 540-231-9292; Fax: 540-231-9070; E-mail: [email protected] relative to recommended adequate intake since 1997 of 200 IU (5 μg) Received October 17, 2013; Accepted October 30, 2013; Published November per day for children and adults to up to age 50 y and 400 -600 IU (10- 04, 2013 15 μg) for adults>50 years of age [22]. Other researchers recommend Citation: Phillips KM, Rasor AS (2013) A Nutritionally Meaningful Increase in 1500-2000 IU/day as the adequate intake in individuals at risk for Vitamin D in Retail Mushrooms is Attainable by Exposure to Sunlight Prior to Consumption. J Nutr Food Sci 3: 236. doi: 10.4172/2155-9600.1000236 vitamin D deficiency [5,23]. The limited natural dietary sources of vitamin D [(primarily fatty fish and fish oils [7]] and increasingly Copyright: © 2013 Phillips KM, et al. This is an open-access article distributed 3 under the terms of the Creative Commons Attribution License, which permits recognized suboptimal vitamin D status of individuals has created a unrestricted use, distribution, and reproduction in any medium, provided the need for alternatives to improve intake [24-26]. Dietary supplements original author and source are credited.

J Nutr Food Sci ISSN: 2155-9600 JNFS, an open access journal Volume 3 • Issue 6 • 1000236 Citation: Phillips KM, Rasor AS (2013) A Nutritionally Meaningful Increase in Vitamin D in Retail Mushrooms is Attainable by Exposure to Sunlight Prior to Consumption. J Nutr Food Sci 3: 236. doi: 10.4172/2155-9600.1000236

Page 2 of 8 in the U.S. retail market cultivated varieties [e.g., common white button, or submaximal UV intensity would result in a nutritionally meaningful crimini, and portabella (Agaricus bisporus)] had nominal vitamin D2 increase in vitamin D content [e.g., ≥ 25% of the DRI (150 IU, 3.75 µg) levels (<20 IU (0.5 µg) per 100 g) and wild grown types (e.g., morel, per 70 g serving]. chanterelle) on average had higher contents [206-212 IU (5.15-5.30 µg) There are limited data on the consistency and magnitude of per 100 g]. The differences were not correlated with ergosterol content increases in vitamin D that can be expected using ambient sun and were likely due to variable exposure to ambient UV. Mushroom 2 exposure, consisting of anecdotal reports [44,45]. A practical protocol producers have taken advantage of the conversion of ergosterol to for exposure of mushrooms to sunlight as a simple and inexpensive vitamin D by UV [29,33,38-41] to generate vitamin D enhanced 2 means to increase vitamin D intake was tested to evaluate the potential mushrooms with commercial scale exposure to UV light. Vitamin for generating a nutritionally significant increase in the vitamin D D enhanced mushrooms are available in the U.S. [e.g., Sun Bella™ content of white button mushrooms using a consumer-based approach. (Monterey Mushrooms, Watsonville, CA); Dole® Vitamin D Portobello mushrooms (Oakshire Mushroom Sales, LLC, Kennett Square, PA)]. The Methods average vitamin D2 content of enhanced portabella mushrooms sampled from U.S. retail markets was 448 IU (11.2 µg) per 100 g compared to only Study design 10 IU (0.25 µg) per 100 g in untreated portabella [37]. A series of experiments on sliced white button mushrooms Controlled studies have shown a large and rapid increase in vitamin (Agaricus bisporus) was conducted in Blacksburg, Virginia (U.S.A.) D in mushrooms exposed to UV light [38,40,42]. For example, Roberts between March 2011 and March 2013 on different days and under et al. [40] reported an increase of >1400 IU/100 g (>35 µg/100 g) in varying conditions that affect UV exposure (e.g., cloud cover, season, white button mushrooms after 8 minutes of UV-B irradiation at 0.5 J/ time of day and duration of exposure), as summarized in Table 1. The 2 2 approach was to use a consistent protocol that would be simple and cm , 1.0 mW/cm . Simon et al. [43] found vitamin D2 increases in sliced white button mushrooms subjected to commercial scale UV treatment practical for consumers to implement, and do repeated trials over a or 2.5 hr sunlight to be similar, going from 1.6 IU (0.4 µg) per 100 g variety of conditions giving a wide range of UV exposure. A total of in the untreated mushrooms to 1200 IU (30 µg) per 100 g for both 16 experiments were done following the uniform protocol described methods of exposure. This content is 840 IU per 70 g serving and 140% below. of the DRI, compared to fortified milk with 100 IU (16.7% DRI) per Samples and sample treatment 8 oz. (240 mL) serving [21]. Therefore it is reasonable to expect that exposure of mushrooms to sunlight for relatively short duration and/ All sample preparation other than sun exposure was performed in

Exposure conditions Exposure time (min.) UV Expt. Date Season Location* (post-exposure Time of day Surface index treatment) 15 30 60 A 21-Mar-2011 Spring Blacksburg, VAb Partly cloudy n.d. 1:37 pm Aluminum pan x B 29-Jun-2011 Summer Blacksburg, VAb Clear 4 - 10 2:45 pm Aluminum pan x Aluminum foil x x White paper on glass pan x C 30-Aug-2011 Blacksburg, VAb Clear 8 - 9 2:17 pm Summer Dark non-stick pan x Aluminum pan x x D 12-Oct-2011 Fall Blacksburg, VAb Overcast 1 - 2 1:46 pm Aluminum foil x x E 6 -7 1:30 pm Portabello, sliced x 6 - 7 1:30 pm Shiitake, sliced x 6 - 7 1:30 pm Aluminum foil Enoki, whole x 4 - 5 3:30 pm White button, whole x 4 - 5 3:30 pm Oyster, sliced x F 18-Oct-2011 Fall Blacksburg, VAc Partly cloudy 0 - 2 5:07 pm Aluminum foil x G 7-Mar-2012 Winter Blacksburg, VAb Clear 2 - 6 2:28 pm Aluminum foil x H 10-Mar-2012 Winter Blacksburg, VAb Clear 8 - 9 10:32 am Aluminum foil x I 6-Apr-2012 Spring Blacksburg, VAc Clear 2 - 7 4:07 pm Aluminum foil x J 7-Apr-2012 Spring Christiansburg, VAd Clear 7 - 9 11:15 am Aluminum foil x K1 4-Jun-2012 Summer Dexter, MIe Mostly clear n.d. 11:00 am Aluminum foil x K2 9-Jun-2012 Summer Corning, NYf Mostly clear n.d. 11:00 am Aluminum foil x K3 10-Jul-2012 Summer Dallas, TXg Mostly cloudy n.d. 11:00 am Aluminum foil x K4 31-Jul-2012 Summer Beltsville, MDh Mostly clear n.d. 10:45 am Aluminum foil x** L 6-Feb-2013 Winter Blacksburg, VAb Clear 4 12:52 pm Aluminum foil x M 25-Feb-2013 Winter Blacksburg, VAb Mostly cloudy 0 - 1 9:51 am Aluminum foil x x N 14-Mar-2013 Spring Blacksburg, VAi Partly cloudy 2 9:09 am Aluminum foil x n.d.= not determined *Latitude, longitude (elevation above sea level; orientation): b37.2264217, -80.4255651 (618m; east); c 37.2264217, -80.4255651 (618m; west); d 37.1438000, -80.4293000 (609m; east); e 42.329171, -83.872428 (277m; south-southeast) ; f 42.138641, -77.056551 (330m; north); g 33.03771, -96.836331 (210m, north- northwest); h39.032348, -76.911002 (42m; east); i 37.2432360, -80.4257903 (645m; southeast) **18 min. Table 1: Summary of parameters for experiments conducted. All experiments except E were with sliced white button mushrooms.

Page 3 of 8 a laboratory with UV-filtered light. For each experiment, mushrooms white button mushrooms, to provide preliminary data on increases in

(12-24 oz, usually 2-3 packages) were purchased locally within 24 vitamin D2 that could be expected in different types of mushrooms. hours of use. Sliced white button and sliced shiitake mushrooms were Mushrooms were locally purchased and treated using the procedure purchased pre-cleaned and used without further preparation. A soft described for sliced white button mushrooms, with exposure on nylon bristled brush was used to remove debris from whole white aluminum foil for 15 minutes on a clear day (17-Oct-2011, UV index button and sliced portabella mushrooms, but no further trimming was 4-5) at 1:30 p.m. in Blacksburg, VA at the same location as for the done. Approximately 1.25 cm was trimmed from the bottom of whole repeated experiments with sliced white button mushrooms. Composites enoki mushrooms and regarded as waste, and no further cleaning was were prepared and analyzed for vitamin D2 as described for sliced white done. Oyster mushrooms were purchased whole (pre-cleaned) and button mushrooms. sliced immediately prior to the experiment. All mushrooms for a given experiment were combined and mixed thoroughly then subdivided Analysis of vitamin D2 into ~225 g quantities for a control (non-exposed) sample and ~225 Vitamin D2 was extracted and analyzed by high performance liquid g mushrooms to be subjected to each treatment. Mushrooms were chromatography (HPLC) at Heartland Assays, Inc. (Ames, IA) using spread on the surface (aluminum foil, aluminum pan, non-stick pan, methodology previously reported [37]. Briefly, mushroom samples or white paper) and placed outdoors in a non-shaded location. The 3 with [ H]-vitamin D3 added as an internal standard were saponified UV index was measured with a UV meter (Oregon Scientific model in methanolic KOH, purified by solid-phase extraction and HPLC to EB612) in all experiments as an estimate of actual UV exposure. The isolate the vitamin D fraction, and vitamin D2 was then separated by control mushrooms for each experiment were homogenized in liquid reverse-phase HPLC with UV detection at 265 nm and quantified based nitrogen using a Robot Coupe® Blixer BX6V food processor (Robot 3 on the ratio of sample peak area to [ H]-vitamin D3 ratio relative to an Coupe USA, Inc., Jackson, MS) at the time the treated mushrooms external standard curve from analysis of vitamin D2 standards spiked began exposure. At the end of the treatment period the exposed the with an equivalent amount of internal standard. mushrooms were immediately homogenized in the same manner. All subsamples were dispensed into 2-oz wide-mouth straight-sided jars Quality control and statistical analyses ® (Qorpak , Bridgeville, PA), surrounded with foil, sealed with tape, and Each composite was assayed in blinded duplicate or triplicate, and a stored at -60°C in darkness until analyzed. blinded sample of a mushroom control composite (“Mushroom CC”, a An experiment was done to compare results from exposure of mixture of portabella and vitamin D enhanced portabella mushrooms) sliced white button mushrooms placed on different surfaces [aluminum was included in each analytical batch, with the analyst blinded to the foil, an aluminum pan, a dark non-stick pan, and white paper (Table 1, identity of replicate samples. The Mushroom CC had been previously experiment C)]. Based on the results of this experiment, aluminum foil analyzed and had an established confidence interval for vitamin 2D [37] (shiny side up) was used in the suggested protocol that was tested in the and was the same control used in previous reports [37,43]. The value for full series of experiments. The effect of storage on the vitamin D content the Mushroom CC in each batch was compared to established tolerance of exposed mushrooms was also evaluated in one experiment (Table limits, and if it fell outside the range all samples in the batch were rerun. 1, experiment A) by placing a portion of treated mushrooms into an Moisture in each composite was determined by vacuum drying to a aluminum foil covered bowl in the refrigerator (2-5°C) overnight (24 constant weight at 60°C and 25 mm Hg [46]. hr) prior to homogenization and analysis. The outcome variable was increase in vitamin 2D per 70 g (1 cup) serving, calculated as the assayed vitamin D content of treated Treatment of mushrooms by consumers at different 2 mushrooms minus the mean vitamin D content of corresponding geographic locations 2 untreated mushrooms), when there was a statistically significant After conducting experiments under different conditions of UV difference in the assayed mean for the treated and untreated mushrooms. exposure in the same location in Blacksburg, trials were implemented Means and standard deviations were calculated using Microsoft® Office by consumers at two homes within the same local area and at four Excel (Professional edition 2010; Microsoft Corporation, Redmond, homes in other geographic locations (Ann Arbor, MI; Corning, WA). Analysis of variance (α=0.05) and pairwise comparison of NY; Dallas, TX; Beltsville, MD) (Table 1, experiments K1-K4). Each means using the Tukey test [47] with a 95% confidence interval were individual was given a procedure to follow which replicated the method performed using XLSTAT (version 2012.4.03; Addinsoft, New York, used in the experiments in Blacksburg. Sliced white button mushrooms NY). The relationship between UV index and increase in vitamin D2 (12-16 oz.) were purchased locally by the participants during June- was evaluated through bivariate regression analysis of exposure time x July 2012. After setting aside half of the mushrooms as a non-exposed UV index as the independent variable, using the JMP 10.0.2 software control, each participant exposed the mushrooms between 10:30 and package (SAS Institute Inc., Cary, NC). 11:30 a.m. local time for 18-30 min. on aluminum foil. The control and treated mushrooms were frozen overnight in separate Rubbermaid® Results containers in home freezers, then shipped to Blacksburg the next day Quality control on ice packs via priority express overnight delivery. Composites were prepared and analyzed for moisture and vitamin D2 as described for the The vitamin 2D concentration in the Mushroom CC included in each on-site experiments. batch of samples assayed (n=10) ranged from 244-296 IU/100 g [mean, 273; median, 272; relative standard deviation (RSD), 6.2%]. All values Evaluation of different mushroom types fell within the mean ± 2*standard deviation of previous results obtained In a separate experiment (E, Table 1), whole white button, sliced (Figure 1). The Mushroom CC samples were stored in the same type of portabella, sliced shiitake (Lentinus edodes), sliced oyster (Pleurotus containers and under the same conditions as the mushroom samples in ostreatus), and whole enoki (Flammulina velutipes) mushrooms were this study prior to analysis, and the consistency of the assayed vitamin exposed at the same time and following the same protocol as for sliced D2 concentration over a 3.8 year period demonstrates stability of the

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500 Figure 2 illustrates these increases in vitamin D2 for sliced white button mushrooms. Results of means comparisons are given in Table 3. 450 For experiments in the Blacksburg area under varying conditions of UV 400 exposure (experiments A-J and L-N, Table 1), increases of <25% of the 350 DRI occurred only in experiments N, F, and M (7.3, 24, and 78 IU/70 300 /100 g) g, respectively) after 15 minutes exposure on partly to mostly cloudy (IU 2 250 days before 10:00 a.m. (N and M) or after 5 p.m. (F), as indicated in 200 Table 1. With 15 min. additional exposure, however, the mushrooms Vitamin D 150 with a level of 78 IU/70 g in experiment M (mostly cloudy day, winter,

100 9:51 a.m., UV index 0-1) had a total increase of 168 IU/70 g (28% DRI). 50 Considering all other treatments that resulted in >150 IU/70 g 0 28-Dec-08 16-Jul-09 1-Feb-10 20-Aug-10 8-Mar-11 24-Sep-11 11-Apr-12 28-Oct-12 16-May-13 increase in vitamin D2, for samples exposed during the mid-day (9:30 Analysis Date a.m.-3:30 p.m.) on clear days, regardless of season (experiments B, Values from this study Values from assays reported in Phillips et al. (37) Reported in Simon et al. (43) Other analyses C, G, H, I, L), vitamin D2 rose 157-754 IU/70 g (26-126%DRI) after All results prior to this study: Mean (12% relative standard deviation) Mean ± 2*standard deviation 15 minutes, and (in experiment C) another 271-438 IU/70 g after Figure 1: Results for samples of Mushroom Control Composite analyzed in an additional 15 minutes. On an overcast day (fall season, mid-day; this study compared to previous results, over a 3.8 year period. experiment D) vitamin D2 increased by 178 IU/70 g (30%DRI) after 15 minutes exposure, and by 510 IU after 1 hour, which was within the

a range in mushrooms exposed 15 min. on clear days. Experiment (Table 1) Vit D2 (IU/70 g serving) A 27.7 ± 2.5 Table 4 summarizes the change in vitamin D2 in sliced white B 11.2b button mushrooms placed on different surfaces (aluminum pan, C 10.5 ± 4.9 aluminum foil (shiny side up), non-stick pan, or white paper) and D 3.4 ± 4.1 exposed to sunlight mid-day with clear skies (Table 1, experiment C). F 3.8 ± 0.5 After 15 minutes, vitamin 2D increased from only 10.5 IU/70 g in the unexposed mushrooms to 661-765 IU/70 g. The change of 651 IU/100 G < 7 g in mushrooms exposed on white paper was slightly lower than in the H < 7 mushrooms treated on the other surfaces (p=0.038), with no measured I < 7 difference between the others. The change was dramatic in all cases, taking the mushrooms from providing < 2% of the RDA to >110% J < 7 per serving. An additional vitamin D2 increase of 271-439 IU/70 g K1 < 4 resulted after a total of 30 minutes exposure for the aluminum pan and K2 < 4 aluminum foil surfaces, yielding 1035-1153 IU/70 g (172-192%DRI).

K3 < 4 In the comparison of the vitamin D2 content of treated mushrooms K4 11.7 ± 2.9 immediately after exposure and after storage overnight in the refrigerator L 23.0 ± 21.0 (24 hours), the concentration did not change significantly from the M 12.8 ± 8.0 level of 738 IU/70 g immediately after exposure (p>0.05). This is not N 8.0 ± 1.3 unexpected, given the <10% loss of vitamin D2 in cooked mushrooms aMean ± standard deviation 1400 bnot applicable (n=1)

Table 2: Vitamin D2 content of untreated sliced white button mushrooms in 1200 experiments described in Table 1. C

C3 1000 nutrient in the samples. The analytical precision achieved is sufficient K2 g 70 g serving) to allow confidence that data from separate analytical batches, that also 800 3 2 C (IU per(IU C K4* 3 2 C K1 A contained samples from different experiments, did not differ due to C1 B3 significant analytical variability. Further, the results for the Mushroom 600 D L CC were consistent with those from analyses done as part of other 400 K3 J published studies on vitamin D in mushrooms [37,43], allowing D Vitamin in Increase I G H 200 D confidence in comparing results for samples among these studies. M I

M NF Sliced white button mushrooms exposed under varying 0 0 - 2 3 - 5 7 - 10 conditions UV Index Exposure time: 15 minutes (*18 min.) 30 min. 60 min. DRI - - - - 25% DRI - - - - 10% DRI The vitamin D content of unexposed sliced white button 2 Figure 2: Increase in vitamin D2 in sliced white button mushrooms exposed to mushrooms in all experiments (n=16) was <30 IU/70 g (< 5% DRI), sunlight under varying conditions of UV intensity (mean ± standard deviation; 1 IU = 0.025 µg). Vitamin D2 in the control (unexposed) mushrooms was < 30 with a range of <3 to 28 IU/70 g and a median of <7 IU/70 g (< 1.2% IU/70 g (Table 3) and differed significantly from the corresponding exposed DRI) (see Table 2). The difference in the mean assayed vitamin D mushrooms in all cases (α=0.05). Letters on data points correspond to 2 experiments in Table 1. Data points with no superscript were for mushrooms concentration in the untreated and treated mushrooms was statistically placed on aluminum foil, and superscripts indicate mushrooms exposed on significant (p<0.05) in all experiments and exposure times shown in 1white paper, 2dark non-stick pan, and 3aluminum pan. DRI= Dietary Reference Table 1. Intake (20). See Table 3 for results of means comparisons.

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Experimenta LS means Groups C (30 min) 1142 A C* (30 min) 1025 A B

K2 903 B

C* (15 min) 754 C

C*** 740 C

A* (60 min) 710 C

C (15 min) 704 C

K4 703 C

K1 695 C

C** 651 C

B* 611 CD

D (60 min) 512 DE

L 427 EF

K3 387 EFG

J 365 FGH

I (30 min) 298 FGHI

G 252 GHI

H 233 HI

D (15 min) 181 IJ

M (30 min) 167 IJK

I (15 min) 155 IJK

M (15 min) 78 JKL

F 24.3 KL N 7.3 L

Table 3: Results of least square (LS) means comparison of increases in vitamin D2 (IU/70g) in mushrooms exposed under different conditions (Table 1 and Figure 2). Increases in vitamin D for experiments denoted by same capital letter for Group do not differ significantly (α=0.05). All treatments were done on an aluminum foil surface except those marked with * (aluminum pan), ** (white paper on glass pan), and *** (dark non-stick pan).

Increase in Vitamin D2 (per 70 g serving) Surface Exposure time (min.) IUa Percent of DRIb Aluminum pan 15 754 ± 27A 126 30 1025 ± 21 B 171 Aluminum foil 15 704 ± 22 A 117 30 1142 ± 73 B 190 Dark non-stick pan 15 740 ± 49 A 123 White paper on glass pan 15 651 ± 33 A 108 aMean ± standard deviation of 2 or 3 replicate analyses. Means that differ significantly (α=0.05) are indicated by different superscripts. 1 IU=5 µg bDietary Reference Intake, 600 IU/day [20].

Table 4: Increase in vitamin D2 in sliced white button mushrooms placed on different surfaces and exposed to sunlight on a clear day (Experiment C, Table 1). Vitamin D2 in the control (unexposed) mushrooms was 10.5 ± 4.9 IU per 70 g serving, and all increases were statistically significant (p<0.0001). and no decrease after 9 months storage at -20°C demonstrated by constraints. However, a wide range of ultimate UV dose received by Mattila et al. [48] for chanterelle mushrooms. treated mushrooms was achieved using variation in parameters that were possible to control (Table 1). The regression equation for increase Prediction of increase in vitamin D 2 in vitamin D2 IU per 70 g serving (ΔD2) as a function of UV index and UV light causes the conversion of ergosterol to vitamin D , exposure time (minutes), assuming no interaction between UV index 2 and exposure time and using data from experiments conducted in the and increases in vitamin D in mushrooms exposed to UV light are 2 Blacksburg area locations using aluminum foil (experiments C-J and related to the UV dose [40]. Parameters that affect UV intensity L-N), was: (including latitude, ozone and smog, elevation) and that would vary ∆=D 3.10 + 3.92*( UV index *exposure time) in generalized implementation of consumer-initiated treatment of 2 mushrooms were not possible to capture in this study due to practical The R-square coefficient was 0.74, suggesting that UV index and

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time explain 74% of the change in vitamin D2. In turn, this suggests a 1000 reasonable degree of confidence in using these parameters to predict D 900 C the increase in vitamin D2 in any particular trial. Theoretically, the 800 variation in UV index reported in the literature, or measurement of the 700 UV index at a particular location where mushrooms are exposed could 600 B C be used to predict outcomes at wide-ranging locations, times of day, serving)g /70 500 (IU seasons, and ambient conditions. While the UV index is not an absolute 2 400 measure, the data and specification of equipment used to measure “UV 300 index” in this study provide a basis for generalizing results. Vitamin D 200 A 100 It should be noted that practical exposure times, from a consumer 0 perspective, were chosen based on the goal of this work. Also, exposure White button, Portabella, sliced Shiitake, sliced Oyster, sliced Enoki, whole whole DRI in full sun for longer than 30 minutes could be expected to affect the Unexposed Exposed 25% DRI quality of the fresh mushrooms. For 60 min. exposure times, some 10% DRI withering and darkening of mushrooms was observed (experiment B). Figure 4: Change in vitamin D2 content of different types of mushrooms exposed to sunlight for 15 minutes on a clear day (mean ± standard deviation). Mushrooms treated by consumers at different geographic Different capital letters indicate means that different significantly (α=0.05). locations DRI=Dietary Reference Intake [20]. In the trials completed by consumers at different locations (Table Figure 4 shows the change in vitamin D2 content of whole white 1, experiments K1, K2, K3, K4) in which mushrooms were placed button, sliced shiitake, sliced oyster, and whole enoki mushrooms in the sun starting between 10:45 and 11:00 a.m. local time, vitamin exposed to sunlight for 15 minutes mid-day, with clear skies (Table 1, D2 increased by 367-905 IU per 70 g serving (61-151%DRI) after 30 experiment E). Whereas the vitamin D2 content of all of the unexposed min. (K1, K2, K3) or 18 min. (experiment K4). The UV index was not mushrooms was low (< 7 to 18.2 IU/70 g), the level increased after measured by consumers, but assuming UV indices of 7 and 3 for the treatment in all types. The largest gains were in oyster and enoki (832 reported “mostly clear” and “mostly cloudy” conditions, the predicted and 886 IU/70 g, respectively). Lower but still dramatic increases increase in vitamin D based on the regression analysis of results from 2 resulted in portabella and shiitake (472 and 529 IU/70 g, respectively). local experiments was calculated. As illustrated in Figure 3, in all cases In whole white button mushrooms the increase was 154 IU/70 g, which but experiment K4, the increase was within 10% of the predicted was lower than in the other mushroom types and the range of 424- amount, while in experiment K4 the increase exceeded what was 704 IU/70 g in sliced white button mushrooms exposed at the same predicted. Although these analyses are preliminary, they suggest it might location under similar conditions (experiments B, C, H, L; Table 1 and be possible to predict the increase in vitamin D2 in a particular location by estimating the UV index at that location relative to Blacksburg, Figure 2). The lower increase is consistent with studies by Jasinghe VA. A number of articles on monitoring and prediction of UV index and Perera [61] that showed using experiments with controlled UV and relative UV exposure at different latitudes and relative to other exposure that orienting mushrooms with the gills facing the UV source factors have been published [49-59]. Similarly, models for estimating resulted in higher vitamin D levels, given the higher surface area of cutaneous UV exposure at different geographic locations as it relates to gills exposed in the sliced mushrooms. Interestingly, however, in whole enoki mushrooms the vitamin D increase was substantially higher than production of vitamin D3 [60] might prove useful in generalizing the in all other types of sliced mushrooms except oyster. It is important results in this study for vitamin D2 production in mushrooms exposed to ambient UV at different locations. to note, however, that even in the whole white button mushrooms just 15 minutes sun exposure resulted in a 14.7% increase in the DRI Other mushroom types contributed by a 70 g serving, which is three times the vitamin D provided by 8 oz. of fortified milk. 1000

900 Conclusion 800 Exposure of sliced white button mushrooms to sunlight proved to 700 (IU /70g )

2 be a simple, reliable, and convenient method by which consumers could 600 increase vitamin D intake. Treatment for as little as 15 minutes on a 500

vi ta mi n D 400 clear or partly cloudy day consistently increased vitamin D2 by at least

300 25% of the DRI (150 IU) per 70 g serving, and more than 100% of the

Increase in Increase 200 DRI (> 600 IU) in many cases. Figure 5 shows the range in vitamin

100 D2 content of sliced white button mushrooms exposed to sunlight for 0 15 minutes in this study compared to the levels in retail samples of Dexter, MI - Dallas, TX - Corning, NY - Beltsville, MD - Mostly clear (K1) Mostly cloudy Mostly clear (K2) Mostly clear (K4) different types of mushrooms and to the vitamin 3D content of other (K3) DRI foods. Even with low UV exposure (UV index ≤ 2), arising from factors Assayed (mean ± 2*standard error) Predicted (± 10%) 25% DRI 10% DRI such as non-optimal orientation to sun, mostly cloudy and overcast

Figure 3: Increase in vitamin D2 content of sliced white button mushrooms conditions, etc., the vitamin D content increased to be comparable to exposed to sunlight by consumers at different geographic locations compared the level in other fortified foods, and was substantially greater after to predicted increase (3.10 + 3.92*UVindex*exposure time), assuming UV indices of 7 and 3 longer exposure times. Preliminary results also suggested the same for “mostly clear” and “mostly cloudy” conditions, respectively. Letters in effects are possible with other types of mushrooms, with possibly parentheses in legend refer to corresponding experiments in Table 1. DRI= greater effects of UV in oyster and enoki. Even though for practical Dietary Reference Intake [20]. reasons the protocol could be tested at limited geographic locations,

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1000 950 900 850 800 750 700 650 600 550 500 450 400 350 Vitamin (IU/serving) D 300 250 200 150 100 50 0

DRI 25% DRI 10%

Retail mushrooms (37) UV index 4 - 10 UV index ≤ 2 Other Foods (62)

Figure 5: Vitamin D2 content of sliced white button mushrooms exposed to sunlight between 9:30 a.m. and 3:30 p.m. Eastern Standard Time, compared to vitamin D2

in retail samples of different mushroom types [37] (mean ± range), and to vitamin D3 in other foods (mean ± standard error) [62]. DRI=Dietary Reference Intake [20].

UV index and exposure time explained 74% of the changes in vitamin 9. Office of Dietary Supplements (2011) Dietary Supplement Fact Sheet: Vitamin D. National Institutes of Health. D2 in the experiments conducted and provides a basis to estimate effects at other geographic locations and conditions (e.g., higher or 10. Pludowski P, Holick MF, Pilz S, Wagner CL, Hollis BW, et al. (2013) Vitamin lower latitudes, higher elevations, different ozone levels) based on UV D effects on musculoskeletal health, immunity, autoimmunity, cardiovascular disease, cancer, fertility, pregnancy, dementia and mortality- a review of recent index. An investment of as little as 15 minutes could provide a low-cost evidence. Autoimmun Rev 12: 976-978. and convenient consumer-based strategy to increase dietary vitamin D 11. Roupas P, Keogh J, Noakes M, Margetts C, Taylor P (2012) The role of edible intake. mushrooms in health: evaluation of the evidence. J Functional Foods 4: 687- Acknowledgements 709. This work was conducted as part of cooperative agreements #59-1235- 12. Urashima M, Segawa T, Okazaki M, Kurihara M, Wada Y, et al. (2010) 7-146 and 58-1235-2-111 between the USDA Nutrient Data Laboratory and Randomized trial of vitamin D supplementation to prevent seasonal influenza A Virginia Tech. The diligent work of Ryan McGinty, Nancy Conley, and Maximilian in schoolchildren. Am J Clin Nutr 91: 1255-1260. Opheim in conducting experiments is acknowledged. We thank Nancy Tietz, Lisa 13. Zhao XY, Feldman D (2001) The role of vitamin D in prostate cancer. Steroids Moore, Matthew Tietz, and David Haytowitz for their assistance in conducting the 66: 293-300. consumer-based trials. 14. Freedman DM, Cahoon EK, Rajaraman P, Major JM, Doody MM, et al. (2013) References Sunlight and other determinants of circulating 25-hydroxyvitamin D levels in black and white participants in a nationwide U.S. study. Am J Epidemiol 177: 1. Bikle DD (2007) What is new in vitamin D: 2006-2007. Curr Opin Rheumatol 180-192. 19: 383-388. 15. Holick MF (2007) Vitamin D deficiency. N Engl J Med 357: 266-281. 2. Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B (2006) Estimation of optimal serum concentrations of 25-hydroxyvitamin D for 16. Battault S, Whiting SJ, Peltier SL, Sadrin S, Gerber G, et al. (2013) Vitamin multiple health outcomes. Am J Clin Nutr 84: 18-28. D metabolism, functions and needs: from science to health claims. Eur J Nutr 52: 429-441. 3. de Borst MH, de Boer RA, Stolk RP, Slaets JP, Wolffenbuttel BH, et al. (2011) Vitamin D deficiency: universal risk factor for multifactorial diseases? Curr Drug 17. Holick MF (2009) Vitamin D status: measurement, interpretation, and clinical Targets 12: 97-106. application. Ann Epidemiol 19: 73-78.

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